Carbohydrate Analysis of Glycoproteins on Blots Using the Immunological System: Digoxigenin/Anti-Digoxigenin Antibody

Abstract

By introducing the steroid hapten digoxigenin specifically into sugars, a sensitive detection system for glycoproteins on blots has been developed. Digoxigenin is bound to sugars either as digoxigenin-hydrazide after oxidation to aldehydes, or as lectin conjugates. Digoxigenin is then detected with a high-affinity anti-digoxigenin antibody, conjugated with alkaline phosphatase or peroxidase. Besides of the general labeling of the carbohydrates, the reaction with digoxigenin-hydrazide can further be made specific for the detection ofsialic acids or terminal galactose. By using digoxigenin labeled lectins with defined and narrow binding specificity for probing of the glycoproteins, it is possible to obtain substantial informations on the glycan structures. The high resolution power of electrophoretical separation systems is widely used for analyzing complex protein mixtures. The combination of these techniques with the sensitive digoxigenin based labeling and detection system allows the rapid analysis of small amounts of glycoproteins, which can be further refined by including exo- and endoglycosidase digestions as specific tools.